WO 2008/148519 PCT/EP2008/004413 Mammalian expression vector with a highly efficient secretory signal sequence The present invention relates to mammalian cell based expression systems and the expression and secretion of recombinant proteins by using secretory signal peptides. The 5 present invention also relates to an expression cassette useful for the secretion of a heterologous gene from a mammalian cell, in particular a CHO cell. The present invention is also directed to methods of secreting a heterologous protein from mammalian cells such as CHO cells and methods for the production of secreted recombinant proteins in mammalian host cells. 10 Recombinant polypeptides for medical, research and veterinary applications such as antibodies are produced using a wide variety of genetically engineered organisms that include prokaryotic and eukaryotic cells. However, a lot of these proteins are glycoproteins requiring post-translational modifications. Thus, prokaryotic host cells such as bacterial 15 cells are not suitable. For this reason other protein expression systems were developed using the cells of higher eukaryotes, e.g. insect cells or mammalian cells. Viral expression systems can produce recombinant proteins both in insects and mouse cell lines but suffer from several serious drawbacks, in particular that purification of recombinant proteins from virus infected systems is very problematic. 20 A major problem in biotechnology exists in the production and recovery of recombinant polypeptides that are not readily secreted such as intracellular proteins or protein subunits, from genetically engineered organisms. Often these intracellular proteins or protein subunits can be expressed at only moderate levels inside a cell and their purification must 25 first include steps to lyse the cells, followed by several procedures to isolate the desired polypeptides from the other intracellular proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. Whenever possible, secretion is the preferred 30 strategy since it permits easy and efficient purification from the extracellular medium. In addition, the secretory production of recombinant proteins has the advantage that proteolytic degradation may be avoided and that there is a better chance of correct protein folding. Successful protein secretion requires effective translocation of the protein across CONFIRMATION COPY WO 2008/148519 PCT/EP2008/004413 2 the endoplasmatic reticulum or plasma membrane. Proteins that are secreted from a cell through a cell membrane are generally produced within the cell in a precursor form, referred to as a "preprotein". This "preprotein" form includes an additional peptide sequence at the amino-terminus which is required to target the nascent peptide chain to the 5 endoplasmatic reticulum to enable its entry into the secretory pathway. This additional peptide sequence is referred to as a signal sequence. However, it is known that secretion frequently does not function to the degree desired, for example because the native signal sequence of the recombinant protein often does not 10 operate well in the host cell. To date, each expression system needs specific tailoring to meet the requirements for each protein product to ensure correct folding, activity and desired yield. Although quite a lot of signal sequences have been identified which might be useful for the secretion of particular recombinant proteins, there is still a need in the art for additional signal sequences that can promote efficient secretion of recombinant proteins, in 15 particular immunoglobulins, in mammalian host cells. Thus, the technical problem underlying the present invention is to provide a method to efficiently secrete non-secretion competent polypeptides, in particular antibody chains from a eukaryotic host cell such as a Chinese Hamster Ovary (CHO) cell. 20 The present invention solves this technical problem by providing an expression cassette for the secretion of a heterologous protein from a mammalian host cell, in particular a CHO cell comprising a promoter, functionally linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, 25 wherein the DNA sequence encoding the signal peptide is selected from the group consisting from SEQ ID No. 2, SEQ No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No.10 or a DNA sequence encoding an amino acid sequence depicted in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or SEQ ID No. 9. 30 It was found by the inventors of the present invention that the signal sequences employed in the expression cassettes provide for a proper processing and an efficient secretion of operably linked polypeptide sequences in mammalian host cells. As an initial screen, nineteen different signal sequences have been tested that were derived from different WO 2008/148519 PCT/EP2008/004413 3 secreted proteins of different species to the CHO cells routinely used for recombinant protein production whereby only 5 of them resulted in an improved secretion of the polypeptides tested. Thus, the experiments, demonstrated that it is generally unpredictable whether a signal sequence from one species would be functional in another species. 5 Two of the five signal sequences, namely V19 (SEQ ID No. 9) and V17 (SEQ ID No. 7), resulted in cell lines with a particularly strong increase in mean antibody concentration over control cell lines. 10 On the basis of the amino acid sequence of signal peptide V19 the following consensus amino acid sequence of a signal peptide was derived: MMRP[hydrophobic amino acid]nTSALA. On the basis of the amino acid sequence of signal peptide V17 the following consensus amino acid sequence of a signal peptide was derived: MKT[hydrophobic amino acid]nCATVHC. 15 Thus, the present invention solves the technical problem also by providing a signal sequence with the general amino acid sequence MMRP[hydrophobic amino acid]nTSALA and also a signal sequence with the general amino acid sequence MKT[hydrophobic amino acid]nCATVHC. Both signal sequences provide for an efficient secretion of operably 20 linked polypeptide sequences, e.g. antibody chains, in mammalian host cells such as CHO cells. Preferably the hydrophobic amino acid in the central region is selected from the group consisting of alanine (Ala or A), isoleucine (Ile or I), leucine (Leu or L), phenylalanine (Phe or F), methionine (Met or M) and valine (Val or V). Ala has a hydropathy index of 1.8. Ile has a hydropathy index of 4.5. Leu has a hydropathy index of 25 3.8. Phe has a hydropathy index of 2.8. Met has a hydropathy index of 1.9. Val has a hydropathy index of 4.2. Preferably the central stretch of hydrophobic amino acids consists especially of Leu (L) with some occurrence of Val, Ala, Phe and Ile. Preferably the central hydrophobic stretch has a length of 4 to 9 amino acid residues. Preferably the central hydrophobic region comprises 4 - 16 amino acid residues, particularly preferred 4-9 30 residues. The present invention also relates to an expression cassette for the secretion of a heterologous protein from a mammalian host cell comprising a promoter, functionally WO 2008/148519 PCT/EP2008/004413 4 linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, wherein the DNA sequence encoding the signal peptide is selected from a DNA sequence encoding the amino acid sequence MMRP[hydrophobic amino acid]nTSALA or a DNA sequence encoding the amino acid 5 sequence MKT[hydrophobic amino acid]nCATVHC whereby n is an integer of 4 to 16 and the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, phenylalanine, methionine and valine. In a particularly preferred embodiment the DNA sequence encoding the signal sequence is 10 selected from SEQ ID No. 8 or SEQ ID No.10 or a DNA sequence encoding the amino acid sequence depicted in SEQ ID No. 7 or SEQ ID No. 9. The present invention also relates to an expression cassette for the secretion of a heterologous protein from a mammalian host cell comprising a promoter, functionally 15 linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, wherein the DNA sequence encoding the signal peptide is selected from the group consisting from SEQ ID No. 2, SEQ No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No.10 or a DNA sequence encoding an amino acid sequence depicted in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or SEQ ID No. 9 20 In the context of the present invention an "expression cassette" is made up of one or more genes to be expressed and sequences controlling their expression such as a promoter/enhancer sequence, including any combination of cis-acting transcriptional control elements. The sequences controlling the expression of the gene, i.e. its transcription 25 and the translation of the transcription product, are commonly referred to as regulatory unit. Most parts of the regulatory unit are located upstream of coding sequence of the heterologous gene and are operably linked thereto. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site. The regulatory unit of the invention is either directly linked to the gene to be expressed, i.e. 30 transcription unit, or is separated therefrom by intervening DNA such as for example by the 5'-untranslated region of the heterologous gene. Preferably the expression cassette is flanked by one or more suitable restriction sites in order to enable the insertion of the expression cassette into a vector and/or its excision from a vector. Thus, the expression WO 2008/148519 PCT/EP2008/004413 5 cassette according to the present invention can be used for the construction of an expression vector, in particular a mammalian expression vector. In the context of the invention the terms "heterologous coding sequence", "heterologous 5 gene sequence", "heterologous gene", "recombinant gene" or "gene of interest" are used interchangeably. These terms refer to a DNA sequence that codes for a recombinant or heterologous protein product that is sought to be expressed in the mammalian cell and harvested in high amount. The product of the gene can be a protein or polypeptide, but also a peptide. The heterologous gene sequence is naturally not present in the host cell and is 10 derived from an organism of a different species. The product of the gene may be any protein of interest, e.g. a therapeutic protein such as an interleukin or an enzyme or a subunit of a multimeric protein such as an antibody or a fragment thereof. Preferably, the protein is an antibody or engineered antibody or a 15 fragment thereof, most preferably it is an Immunoglobulin G (IgG) antibody. According to the present invention the terms "signal peptide" or "signal sequence" are used interchangeably and refer to a short continuous stretch of amino acids residues at the amino-terminus of secreted and membrane-bound proteins. The signal peptide targets the 20 protein to the secretory pathway and is cleaved from the nascent chain once translocated in the reticulum endoplasmatic membrane. The signal peptide consists of three regions: an amino-terminal polar region (N region), where frequently positive charged amino acid residues are observed, a central hydrophobic region (H region) of 7-8 amino acid residues and a carboxy-terminal region (C region) that includes the cleavage site. The cleavage of 25 the signal peptide from the mature protein occurs at this cleavage site. A "promoter" is defined as a regulatory DNA sequence generally located upstream of a gene that mediates the initiation of transcription by directing RNA polymerase to bind to DNA and initiating RNA synthesis. 30 The terms "functionally linked" and "operably linked" are used interchangeably and refer to a functional relationship between two or more DNA segments, in particular gene sequences to be expressed and those sequences controlling their expression. For example, a WO 2008/148519 PCT/EP2008/004413 6 promoter/enhancer sequence, including any combination of cis-acting transcriptional control elements is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Promoter regulatory sequences that are operably linked to the transcribed gene sequence 5 are physically contigous to the transcribed sequence. Another aspect of the present invention relates to a mammalian expression vector comprising a promoter functionally linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, wherein the 10 DNA sequence encoding the signal peptide is selected from a DNA sequence encoding the amino acid sequence MMRP [hydrophobic amino acid]n TSALA or a DNA sequence encoding the amino acid sequence MKT [hydrophobic amino acid]n CATVHC whereby n is an integer of 4 to 16 and the hydrophobic amino acid is A, I, L, M, F or V. Preferably the DNA sequence encoding the signal sequence is selected from SEQ ID No. 8 or SEQ ID 15 No.10 or a DNA sequence encoding an amino acid sequence depicted in SEQ ID No. 7 or SEQ ID No. 9. The present invention also relates to a mammalian expression vector comprising a promoter, functionally linked to a DNA sequence encoding a signal peptide which is linked 20 in frame to a DNA sequence encoding a heterologous protein, wherein the DNA sequence encoding the signal peptide is selected from the group consisting from SEQ ID No. 2, SEQ No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No.10 or a DNA sequence encoding an amino acid sequence depicted in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or SEQ ID No. 9. 25 Thus, the present invention also relates to mammalian expression vectors comprising an expression cassette for the secretion of a heterologous protein according to the invention. In the context of the present invention a "mammalian expression vector" is a, preferably 30 isolated and purified, DNA molecule which upon transfection into an appropriate mammalian host cell provides for a high-level expression of a recombinant gene product within the host cell. In addition to the DNA sequence coding for the recombinant or heterologous gene product the expression vector comprises regulatory DNA sequences that WO 2008/148519 PCT/EP2008/004413 7 are required for an efficient transcription of the DNA coding sequence into mRNA and for an efficient translation of the mRNAs into proteins in the host cell line. In a preferred embodiment of the invention the mammalian expression vector comprises at 5 least two separate transcription units. An expression vector with two separate transcription units is also referred to as a double-gene vector. An example thereof is a vector, in which the first first transcription unit encodes the heavy chain of an antibody or a fragment thereof and the second transcription unit encodes the light chain of an antibody. Another example is a double-gene vector, in which the two transcription units encode two different 10 subunits of a protein such as an enzyme. However, it is also possible that the inventive expression vector comprises more than two separate transcription units, for example three, four or even more separate transcription units each of which comprises a different nucleotide sequence encoding a different polypeptide chain. An example therefore is a vector with four separate transcription units, each of which contains a different nucleotide 15 sequence encoding one subunit of an enzyme consisting of four different subunits. Preferably, the mammalian expression vectors of the present invention further contain at least one expressible marker selectable in animal cells. Any selection marker commonly employed such as thymidine kinase (tk), dihydrofolate reductase (DHFR) or glutamine 20 synthetase (GS) may be used. In a particular preferred embodiment, the mammalian expression vector contains a GS selection marker (Bebbington et al., 1992, High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable 25 selectable marker, Bio/Technology 10:169-175; Cockett et al., 1990, High level expression of tissue inhibitor of metalloproteinases in Chinese Hamster Ovary (CHO) cells using Glutamine synthetase gene amplification, Bio/Technology 8: 662-667). The GS-system is one of only two systems that are of particular importance for the production of therapeutic proteins. In comparison to the dihydrofolate reductase (DHFR) system, the GS system 30 offers a large time advantage during development because highly productive cell lines can often be created from the initial tranfectant thus avoiding the need for multiple rounds of selection in the presence of increasing concentrations of selective agent in order to achieve gene amplification (Brown et al., 1992, Process development for the production of WO 2008/148519 PCT/EP2008/004413 8 recombinant antibodies using the glutamine synthetase (GS) system, Cytotechnology 9:231-236). Preferably, the expression vectors of the invention also contain a limited number of useful 5 restriction sites for insertion of the expression cassette for the secretion of a heterologous protein of the present invention. Where used in particular for transient/episomal expression only, the expression vectors of the invention may further comprise an origin of replication such as origin of Epstein Barr Virus (EBV) or SV40 virus for autonomous replication/episomal maintenance in eukaryotic host cells but may be devoid of a selectable 10 marker. Transient expression in cell lacking relevant factors to facilitate replication of the vector is also possible. A further aspect of the present invention relates to a mammalian host cell containing the mammalian expression vector according to the invention. The terms "host cell' or "host 15 cell line" refer to any cells, in particular mammalian cells, which are capable of growing in culture and expressing a desired protein recombinant product protein. The mammalian host cell can be a human or non-human cell. Preferred examples of the mammalian host cells include, without being restricted to, MRC5 human fibroblasts, 983M 20 human melanoma cells, MDCK canine kidney cells, RF cultured rat lung fibroblasts isolated from Sprague-Dawley rats, B16BL6 murine melanoma cells, P815 murine mastocytoma cells and MT1A2 murine mammary adenocarcinoma cells. In a particular preferred embodiment the mammalian host cell is a Chinese hamster ovary 25 (CHO) cell or cell line (Puck et al., 1958, J. Exp. Med. 108: 945-955). Suitable CHO cell lines include e.g. CHO KI (ATCC CCL-61), CHO pro3-, CHO DG44, CHO P12 or the dhfr- CHO cell line DUK-BII (Chassin et al., PNAS 77, 1980, 4216-4220) or DUXB 11 (Simonsen et al., PNAS 80, 1983, 2495-2499). 30 For introducing the expression vector into an mammalian host cell according to the present invention any transfection technique such as those well-known in the art, e.g. electoporation, calcium phosphate co-precipitation, DEAE-dextran transfection, lipofection, can be employed if appropriate for a given host cell type. It is to be noted that WO 2008/148519 PCT/EP2008/004413 9 the mammalian host cell transfected with the vector of the present invention is to be construed as being a transiently or stably transfected cell line. Thus, according to the present invention the present mammalian expression vector can be maintained episomally or can be stably integrated in the genome of the mammalian host cell. 5 A transient transfection is characterised by non-appliance of any selection pressure for a vector borne selection marker. In transient expression experiments which commonly last 20-50 hours post transfection, the transfected vectors are maintained as episomal elements and are not yet integrated into the genome. That is the transfected DNA, does not usually 10 integrate into the host cell genome. The host cells tend to lose the transfected DNA and overgrow transfected cells in the population upon culture of the transiently transfected cell pool. Therefore expression is strongest in the period immediately following transfection and decreases with time. Preferably, a transient transfectant according to the present invention is understood as a cell that is maintained in cell culture in the absence of 15 selection pressure up to a time of 90 hours post transfection. In a preferred embodiment of the invention the mammalian host cell e.g. the CHO host cell is stably transfected with the mammalian expression vector of the invention. Stable transfection means that newly introduced foreign DNA such as vector DNA is becoming 20 incorporated into genomic DNA, usually by random, non-homologous recombination events. The copy number of the vector DNA and concomitantly the amount of the gene product can be increased by selecting cell lines in which the vector sequences have been amplified after integration into the DNA of the host cell. Therefore, it is possible that such stable integration gives rise, upon exposure to further increases in selection pressure for 25 gene amplification, to double minute chromosomes in CHO cells. Furthermore, a stable transfection may result in loss of vector sequence parts not directly related to expression of the recombinant gene product, such as e.g. bacterial copy number control regions rendered superfluous upon genomic integration. Therefore, a transfected host cell has integrated at least part or different parts of the expression vector into the genome. 30 A further aspect of the present invention relates to a process for the production of a recombinant protein, comprising the steps of a) transfecting a mammalian host cell or host cell line with an expression vector, b) WO 2008/148519 PCT/EP2008/004413 10 culturing the cell under appropriate conditions to enable propagation of the cell and expression of the recombinant protein and secretion of the recombinant protein from the host cell into the medium and c) harvesting the recombinant protein secreted into the medium. 5 Thus the present invention also relates to a method for the production of a recombinant protein, comprising a) transfecting a mammalian host cell or host cell line with an expression vector, b) culturing the cell under appropriate conditions to enable propagation of the cell and expression of the recombinant protein and secretion of the recombinant 10 protein from the host cell into the medium and c) harvesting the recombinant protein produced from the medium. Suitable media and culture methods for mammalian cell lines are well-known in the art, as described in US 5,633,162 for instance. Examples of standard cell culture media for 15 laboratory flask or low density cell culture and being adapted to the needs of particular cell types include, without being restricted to, Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p.
5 19 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, lp.173 ff, 1963), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential medium 20 (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc.53, p288 ff. 1965) or Iscoves' modified DMEM lacking albumin, transferrin and lecithin (Iscoves et al., J. Exp. med. 1, p. 923 ff., 1978). For instance, Ham's F1O or F12 media were specially designed for CHO cell culture. Other media specially adapted to CHO cell culture are described in EP-481 791. It is known that such culture media can be supplemented with fetal bovine 25 serum (FBS, also called fetal calf serum FCS), the latter providing a natural source of a plethora of hormones and growth factors. The cell culture of mammalian cells is nowadays a routine operation well-described in scientific textbooks and manuals, it is covered in detail e.g. in R. Ian Fresney, Culture of Animal cells, a manual, 4* edition, Wiley Liss/N.Y., 2000. 30 In a preferred embodiment of the present invention the cell culture medium used is devoid of fetal calf serum (FCS or FBS), which then is being termed 'serum-free'. Cells in serum free medium generally require insulin and transferrin in a serum-free medium for optimal WO 2008/148519 PCT/EP2008/004413 11 growth. Transferrin may at least partially be substituted by non-peptide chelating agents or siderophores such as tropolone as described in WO 94/02592 or increased levels of a source of an organic iron favorably in conjunction with antioxidants such as vitamin C. Most cell lines require one or more of synthetic growth factors (comprising recombinant 5 polypeptides), including e.g. epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin like growth factors I and II (IGFI, IGFII), etc.. Other classes of factors which may be necessary include: prostaglandins, transport and binding proteins (e.g. ceruloplasmin, high and low density lipoproteins, bovine serum albumin (BSA)), hormones, including steroid-hormones, and fatty acids. Polypeptide factor testing is best 10 done in a stepwise fashion testing new polypeptide factors in the presence of those found to be growth stimulatory. Those growth factors are synthetic or recombinant. There are several methodological approaches well-known in animal cell culture, an exemplary being described in the following. The initial step is to obtain conditions where the cells will survive and/or grow slowly for 3-6 days after transfer from serum-supplemented culture 15 medium. In most cell types, this is at least in part a function of inoculum density. Once the optimal hormone/growth factor/polypeptide supplement is found, the inoculum density required for survival will decrease. In another preferred embodiment, the cell culture medium is protein-free, that is free both 20 of fetal serum and individual protein growth factor supplements or other protein such as recombinant transferrin. In another embodiment the process of the present invention directed to expression and harvest of the recombinant product protein includes a high-density growth of the animal 25 host cells e.g. in an industrial fed-batch bioreactor. Conventional downstream processing may then be applied. Consequently, a high-density growth culture medium has to be employed. Such high-density growth media can usually be supplemented with nutrients such as all amino acids, energy sources such as glucose in the range given above, inorganic salts, vitamins, trace elements (defined as inorganic compounds usually present at final 30 concentrations in the micromolar range), buffers, the four nucleosides or their corresponding nucleotides, antioxidants such as glutathione (reduced), vitamin C and other components such as important membrane lipids, e.g. cholesterol or phosphatidylcholine or lipid precursors, e.g. choline or inositol. A high-density medium will be enriched in most WO 2008/148519 PCT/EP2008/004413 12 or all of these compounds, and will, except for the inorganic salts based on which the osmolarity of the essentially isotonic medium is regulated, comprise them in higher amounts (fortified) than the afore mentioned standard media as can be incurred from GB2251 249 in comparison with RPMI 1640. Preferably, a high-density culture medium 5 according to the present invention is fortified in that all amino acids except for tryptophan are in excess of 75 mg/l culture medium. Preferably, in conjunction with the general amino acid requirement, glutamine and/or asparagine are in excess of 1 g/l, more preferably of 2 g/l of high-density culture medium. In the context of the present invention, high-density cell culture is defined as a population of animal cells having temporarily a density of viable 10 cells of at least or in excess of 105 cells/ml, preferably of at least or in excess of 106 cells/ml, and which population has been continuously grown from a single cell or inoculum of lower viable cell density in a cell culture medium in a constant or increasing culture volume. 15 In a further preferred embodiment the process of the present invention includes a fed-batch culture. A fed-batch culture is a culture system wherein at least glutamine, optionally with one or several other amino acids, preferably glycine, is fed to the cell culture as described in GB2251249 for maintaining their concentration in the medium, apart from controlling glucose concentration by separate feed. More preferably, the feed of glutamine and 20 optionally one or several other amino acids is combined with feeding one or more energy sources such as glucose to the cell culture as described in EP-229 809-A. Feed is usually initiated at 25-60 hours after start of the culture; for instance, it is useful to start feed when cells have reached a density of about 106 cells/ml. It is well known in the art that in cultured animal cells, 'glutaminolysis' (McKeehan et al., 1984, Glutaminolysis in animal 25 cells,in: Carbohydrate Metabolism in Cultured Cells, ed. M.J. Morgan, Plenum Press, New York, pp. 11-150) may become an important source of energy during growth phase. The total glutamine and/or asparagine feed (for substitution of glutamine by asparagine, see Kurano, N. et al., 1990, J. Biotechnology 15, 113-128) is usually in the range from 0.5 to 10 g per 1, preferably from 1 to 2 g per I culture volume; other amino acids that can be 30 present in the feed are from 10 to 300 mg total feed per litre of culture, in particular glycine, lysine, arginine, valine, isoleucine and leucine are usually fed at higher amounts of at least 150 to 200 mg as compared to the other amino acids. The feed can be added as shot-addition or as continuously pumped feed, preferably the feed is almost continuously WO 2008/148519 PCT/EP2008/004413 13 pumped into the bioreactor. It goes without saying that the pH is carefully controlled during fed-batch cultivation in a bioreactor at an approximately physiological pH optimal for a given cell line by addition of base or buffer. When glucose is used as an energy source the total glucose feed is usually from 1 to 10, preferably from 3 to 6 grams per litre 5 of the culture. Apart from inclusion of amino acids, the feed preferably comprises a low amount of choline in the range of 5 to 20 mg per litre of culture. More preferably, such feed of choline is combined with supplementation of ethanolamine essentially as described in US 6,048,728, in particular in combination with feeding glutamine. It goes without saying that upon use of the GS-marker system, lower amounts of glutamine will be 10 required as compared to a non-GS expression system since accumulation of excessive glutamine in addition to the endogenously produced would give rise to ammonia production and concomitant toxicity. For GS, glutamine in the medium or feed is mostly substituted by its equivalents and/or precursors, that is asparagine and/or glutamate. 15 Methods for harvesting, i.e. isolating and/or purifying a given protein from the medium in which the cells had been cultured are well known in the art. Preferred embodiments of the invention are illustrated in the figures. What is shown is: 20 Figure 1 shows the influence of different signal sequences on the concentration of secreted antibody when used in transient transfections. of CHOKISV cells. Antibody concentrations achieved by transient transfection of CHOK1SV cells using vectors employing the variant signal sequences. n=6. 25 Figure 2 shows the influence of different signal sequences on the concentration of secreted antibody when used in stable cell lines in static culture. Boxplots show the range of antibody concentrations achieved by stable transfection of CHOKISV cells using vectors employing the variant signal sequences. Stable GS cell lines were allowed to overgrow for 14 days in 24 well plates at which point antibody concentration was determined by Protein 30 A HPLC. Mean antibody concentrations are shown and those showing a statistically significant increase in mean antibody concentration (p50.05 calculated by ANOVA) are indicated with a *. n= 100.
WO 2008/148519 PCT/EP2008/004413 14 Figure 3 shows the influence of signal sequence V19 on the concentration of secreted antibody when used in a stable cell line in suspension culture. Boxplots show the range of antibody concentrations achieved by stable transfection of CHOK1SV cells using vectors employing either control or V19 signal sequences. Stable GS cell lines were generated and 5 the top 60 evaluated in a fed-batch process designed to mimic laboratory scale bioreactors. Antibody concentration was determined by Protein A HPLC. Mean antibody concentrations are shown, p value calculated by ANOVA. n=60. Figure 4 shows the influence of signal sequence V19 on the concentration of secreted 10 antibody when a stable cell line was cultured in a 10 L Bioreactor. Boxplots show the range of antibody concentrations achieved in 1OL labscale bioreactors using stable cell lines generated using vectors employing either control or V19 signal sequences. Stable GS cell lines were generated and the top 8 evaluated in a fed-batch process. Antibody concentration was determined by Protein A HPLC. Mean antibody concentrations are 15 shown. n=8. The sequence listing shows: SEQ ID No.1 shows the amino acid sequence of the signal peptide used in construct pV12. 20 This signal peptide was derived from Sandfly Yellow related protein and is as follows: mrfffvflaivlfqgihg. The signal peptide of SEQ ID No. 1 is also referred to as signal peptide V12. SEQ ID No.2 shows the nucleic acid sequence encoding the amino acid sequence of signal 25 peptide depicted in SEQ ID No. 2 and is as follows: 5' atgagattctttttcgtgttcctggccatcgtgctgttccagggcatccacg-3'. SEQ ID No. 3 shows the amino acid sequence of the signal peptide used in construct pV14. This signal peptide was derived from Silkworm Fibroin LC and is as follows: 30 mkpiflvllvvtsaya.. The signal peptide of SEQ ID No. 3 is also referred to as signal peptide V14. SEQ ID No. 4 shows the nucleic acid sequence encoding the amino acid sequence of the WO 2008/148519 PCT/EP2008/004413 15 signal peptide depicted in SEQ ID No. 3 and is as follows: 5' atgaagcccatctttctggtgctgctggtcgtgaccagcgcctacgcc-3'. SEQ ID No. 5 shows the amino acid sequence of the signal peptide used in construct pV16. 5 This signal peptide was derived from Snake PLA2 and is as follows: mrtlwimavlllgveg. The signal peptide of SEQ ID No. 5 is also referred to as signal peptide V16. SEQ ID No. 6 shows the nucleic acid sequence encoding the amino acid sequence of the signal peptide depicted in SEQ ID No. 5 and is as follows: 5' 10 atgaggaccctgtggatcatggccgtgctgctgctgggcgtggagggccaggtg-3'. SEQ ID No. 7 shows the amino acid sequence of the signal peptide used in construct pV17. This signal peptide was derived from Cypridina Noctiluca luciferase and is mktlilavalvycatvhc. The signal peptide of SEQ ID No. 7 is also referred to as signal 15 peptide V17. SEQ ID No. 8 shows the nucleic acid sequence encoding the amino acid sequence of the signal peptide depicted in SEQ ID No. 7 and is as follows: 5' atgaaaaccctgatcctggccgtggccctggtgtactgcgccaccgtgcactgc-3'. 20 SEQ ID No 9 shows the amino acid sequence of the signal peptide used in construct pV19. This signal peptide was derived from Pinemoth Fibroin LC and is mmrpivlvllfatsala. The signal peptide of SEQ ID No. 9 is also referred to as signal peptide V19. 25 SEQ ID No. 10 shows he corresponding nucleic acid sequence encoding the amino acid sequence of the signal peptide depicted in SEQ ID No. 9 and is as follows: 5' atgatgaggcccatcgtgctggtgctgctgttcgccacctccgccctggcccaggtg-3'. The present invention is explained in more detail by the following examples.
WO 2008/148519 PCT/EP2008/004413 16 Examples Materials and Methods 5 Cells used CHO cell line CHOKISV: is a variant of the cell line CHO-KI and has been adapted to growth in suspension and protein-free medium. 10 Propagation of CHOKISV cells: CHOKISV cells were routinely propagated in suspension shaker flasks in CD-CHO medium (Invitrogen) supplemented with 6 mM L-glutamine. Seed concentration was 2 x 105 cells/ml, and cells are split every 4 days. Flasks were gassed with 5% CO 2 and 15 incubated at 36.5*C (between 35.5*C and 37.0*C) with orbital shaking at 140 rpm. Transient Transfection: Transient transfections were performed using suspension-growing cells. Cells were counted 20 and distributed onto wells of a 24-well plate at 2.5 x 105 viable cells per well in a DMEM based medium supplemented with 10% serum and 6 mM L-glutamine, and incubated overnight at +36.5 *C. The following day, the conditioned medium was replaced with 1 mL of fresh medium (as above) and the cells incubated for 3 hours at +37 *C. 25 For each transfection, 5 pg of each of the SGVs (HC and LC-SGVs mixed together) or 5 tg of the DGVs were re-suspended in 100 iL transfection medium (OptiMEM, Invitrogen). For positive controls, cells were also transfected with the vector pcB72.3, which encodes heavy chain and light chain genes for an IgG 4 /kappa antibody which serves as a model antibody. A negative control (water only) was also included. 30 For each transfection, 5 pL of Lipofectamine-2000 reagent (Invitrogen) was diluted in 100 iL transfection medium, mixed and left to stand for 5 minute at room temperature. The DNA and diluted Lipofectamine reagent were combined, mixed and further left to stand at WO 2008/148519 PCT/EP2008/004413 17 ambient temperature for 20 minutes. This 200 ptL mixture was then added to a well of the 24-well plate containing the cells, and the cells were incubated for 4 or 10 days at +37 *C. The culture supernatant was collected and clarified by centrifugation prior to assay for presence of antibody by assembly ELISA. 5 Stable transfections for generation of static cultures: Cells used for transfections were grown in cell suspension culture, as detailed before. Cells from growing cultures were centrifuged and washed once in serum-free medium prior to 10 being re-suspended to a concentration of 1.43 x 10 7 cells/mL. A 0.7 mL volume of the cell suspension and 40 pg of plasmid DNA were added to an electroporation cuvette. The cuvette was then placed in the electroporation apparatus and a single pulse of 250 V and 400 ptF was delivered. Following transfection, the cells were distributed into 96-well plates at approximately 2,500 host cells/well (5 x 10 4 /mL), using the non-selective DMEM-based 15 medium supplemented with 10% dFCS. The plates were incubated at 36.5'C (between 35.5*C and 37.0*C) in an atmosphere of 10% CO 2 in air. The day after the transfection, DMEM-based medium supplemented with 10% dFCS/66 gM L-methionine sulphoximine was added to each well (150 gL/well) to give a final L 20 methionine sulphoximine concentration of 50 pM. The plates were monitored to determine when the non-transfected cells died and when foci of transfected cells appeared. Foci of transfected cells became apparent approximately three to four weeks after transfection. All the cell lines examined and progressed further came from wells containing only a single colony. 25 Assessment of Productivity of Cell Lines in Static Culture The 96-well transfection plates were incubated for approximately three weeks to allow colony formation. The resulting colonies were examined microscopically to verify that the 30 colonies were of a suitable size for assay (covering greater than 60% of the bottom of the well), and that only one colony was present in each well. Suitable colonies were transferred to wells of 24-well plates containing 1 mL of selective WO 2008/148519 PCT/EP2008/004413 18 growth medium (DMEM-based medium/10% dFCS/25jiM L-methionine sulphoximine). These cultures were incubated for 14 days at 36.5*C (between 35.5*C and 37.0*C) in an atmosphere of 10% CO 2 in air. The supernatant of each well was harvested and analysed for the concentration of antibody present by the protein-A HLPC method. 5 Assembly ELISA: The antibody concentration of samples was determined using a sandwich ELISA which measures assembled human IgG. This involved capture of samples and standard onto a 96 10 well plate coated with an anti-human Fc antibody. Bound antibody is revealed with an anti human light chain linked to horseradish peroxidase and the chromogenic substrate TMB. Colour development was proportional to the concentration of antibody present in the sample when compared to the standard. 15 Protein A HPLC: The Protein A affinity chromatography method for the measurement of IgG was performed on an Agilent 1100 HPLC. IgG product binds selectively to a Poros Protein A immunodetection column. Non-bound material is washed from the column and the 20 remaining bound antibody is released by decreasing the pH of the solvent. The elution was monitored by absorbance at 280 nm and product was quantified (using Chemstation software) against a generic antibody standard and a correction is made for differences in extinction coefficients. 25 Stable transfections for generation of suspension cultures: CHOKISV host cells were revived from a stock of the CHOKISV cell line. Cells from subsequent growing cultures were centrifuged and washed once in CD-CHO medium prior to being resuspended at a concentration of 1.43 x 107 viable cells/mL.For each transfection, 30 approximately 0.7 mL of the cell suspension and 40 pg of plasmid DNA were added to each electroporation cuvette. Four transfections were prepared using the contents of two electroporation cuvettes. Each electroporation cuvette was placed in the electroporation apparatus and a single pulse of 300 V, 900 pF was delivered. Following transfection, the WO 2008/148519 PCT/EP2008/004413 19 cells from all the required cuvettes were pooled and distributed into 96-well plates at approximately 2,500 host cells/well (0.50 x 10 5 /mL) to 10,000 host cells/well (2.00 x 10 5 /mL), using the medium CD-CHO/phenol red. Phenol red was added only to indicate cell growth. The plates were incubated at 35.5 to 37.0 'C in an atmosphere of 10% v/v CO 2 5 in air. The day after transfection, 150 ptL of the selective medium (CD-CHO/phenol red/66.6 pIM MSX) was added to each well. The final concentration of MSX in each well was 50 pM. The plates were monitored to determine when the non-transfected cells died to leave foci of 10 transfected cells. Foci of transfected cells became apparent three to four weeks after transfection. All the transfectants examined and progressed further came from wells containing only a single colony, as determined by visual assessment. Selection of cell lines for evaluation in static culture: 15 The 96-well transfection plates were incubated for approximately three to four weeks to allow colony formation. The resulting colonies were examined microscopically to verify that they were of a suitable size for assessment (covering greater than 60% of the bottom of the well), and that only one colony was present in each well. The culture supernatant was 20 removed and assayed for antibody using Lonza's ELISA method. The percentage confluence of the well was assessed at the time of sampling. The value obtained by dividing the assay results by percentage confluence was used to rank the cell lines. High ranking cell lines were expanded into 24-well plates in the medium CD-CHO/phenol 25 red/25pM MSX (this medium was used for the entire static culture period). On reaching confluence, this culture was used to inoculate a T25 flask, whilst the remaining culture was re-fed with fresh growth medium and returned to the incubator. When confluence was reached in the T25 flask, this culture was fed with fresh medium to encourage the cells to multilayer and adapt to suspension culture. 30 For those cell lines progressed from 96-well plates, a second assessment of productivity was undertaken. The 24-well plate cultures that had been progressed and re-fed were incubated for a further fourteen days until confluence or low viability was reached ('overgrown'). At WO 2008/148519 PCT/EP2008/004413 20 this point, culture supernatant was collected and the antibody concentration measured using a Protein A HPLC method. Cell lines were ranked according to productivity and high ranking cell lines were progressed to be evaluated in suspension culture. 5 Expansion of cell lines to CDACF suspension culture: Suspension cultures were initiated from confluent T25 flask cultures, using the medium CD-CHO/25 iM MSX. The choice of inoculation cell concentration was dependent on the measured viable cell concentration in the T25 flask. If the viable cell concentration was 10 greater than 0.40 x 106 viable cells/mL, CD-CHO/25 ptM MSX medium was added to give a viable cell concentration of 0.20 x 106 viable cells/mL in a final volume of 5 to 30 mL in a 125 mL shake-flask. If the viable cell concentration was 0.25 to 0.40 x 106 viable cells/mL, CD-CHO/25 pM MSX medium was added to give a cell concentration of 0.15 x 106 viable cells/mL in a final volume of 5 to 30 mL in a 125 mL shake-flask. If the viable cell 15 concentration was less than 0.25 x 10 6 /mL after a maximum of fourteen days in T25 flasks, 10 mL of each culture was automatically progressed into 10 mL of CD-CHO/25 pM MSX medium in 125 mL shake-flasks. After transfer from static to suspension culture, cell lines were serially subcultured in 20 CD-CHO/25 pM MSX medium on a four day subculture regime, until acceptable and reproducible growth characteristics were attained. Cultures were prepared in 125 mL shake-flasks, containing a 30 mL culture volume, with an initial inoculation cell concentration of 0.05 to 0.20 x 106 viable cells/mL. Once the viable concentration on day 4 (day of subculture) was consistently above 0.40 x 106 viable cells/mL, cultures were 25 routinely inoculated at 0.20 x 106 viable cells/mL. Fed-batch shake-flask culture: Cultures of each selected cell line were prepared in 250 mL shake-flasks with 30 mL of cell 30 suspension using the medium CM42/SPE. The cultures were inoculated at 0.20 x 106 viable cells/mL and the headspace of each culture was equilibrated with 5% v/v CO 2 in air. The cultures were incubated at 35.5 to 37.0 *C on a shaking platform at 140 ± 5 rpm until the viable cell concentration, post peak, was less than or equal to 1.00 x 106 viable cells/mL or WO 2008/148519 PCT/EP2008/004413 21 day 15 was reached ('overgrown'). At this point the cultures were harvested. The cell concentration was determined on days 7 and 14 using a Vi-Cell automated cell counter. On day 3, 2.1 mL of SF40 was added as a bolus to each 30 mL culture. A shot of 5 360 pL of the second feed SF41 was applied to the fed-batch cultures on days 8 and 11. Samples of culture supernatant were taken on days 7 and 14 and frozen at -20 ± 5 *C until assayed for assembled antibody by Protein A HPLC. 1OL Bioreactor Culture: 10 Cell lines were evaluated in 1 OL bioreactors. The volume of the sixteen cell line cultures were increased until sufficient volume was obtained to inoculate one 10 litre airlift bioreactor. The volume of inoculum was adjusted to achieve a seed density of approximately 0.2x 106 cells/ml. Bioreactors were run for 15 days using a fed-batch strategy. 15 Samples of culture supernatant were taken and assayed for assembled antibody by Protein A HPLC. Vector Construction Gene optimised DNA sequences encoding the variable region the cB72.3 antibody were 20 synthesised. As part of the synthesis, a nucleic acid sequence encoding a signal sequence chain was added. In total 19 different signal sequences (sequences V1-V19) were used. Light chain sequences were cloned into the pEE12.4 derived expression vector pConPlusKappa which includes Kappa constant region DNA sequence. Heavy chain were cloned into the pEE6.4 derived vector pConPlusIgG4 which includes IgG4 constant region 25 DNA sequence. Double-gene expression vectors were then generated by cloning Not I/Pvu I fragments of the relevant vector together. The sequence of all constructs was confirmed by DNA sequencing. As an initial screen, nineteen constructs along with an appropriate control employing the 30 signal sequences routinely used were transiently transfected into the CHOK1SV host cell line using Lipofectamine-2000 (Invitrogen). The constructs were transfected six times and the antibody concentration in the media determined by ELISA. The assay identified that five of the nineteen constructs resulted in dramatically increased mean antibody WO 2008/148519 PCT/EP2008/004413 22 concentrations over the control with the remaining constructs resulting in either equivalent or reduced antibody concentrations. These constructs were pV12 containing the signal sequence of SEQ ID No. 1 (signal sequence V12), pV14 containing the signal sequence of SEQ ID No. 3 (signal sequence V 14), pV16 containing the signal sequence of SEQ ID No. 5 5 (signal sequence V16), pV17 containing the signal sequence of SEQ ID No. 7 (signal sequence V17), and pV19 containing the signal sequence of SEQ ID No. 9 (signal sequence V 19). The constructs employing the five signal sequences that demonstrated improved expression 10 in the transient transfection system were then used to create stable cell lines in CHOK1SV cells. The antibody concentration in the media from 100 cell lines per construct was determined by Protein A HPLC. The mean antibody concentration from the control cell lines was 89 mg/L. Two of the five signal sequences resulted in cell lines with a statistically significant increase in mean antibody concentration over control cell lines with 15 sequence V17 (SEQ ID No. 7) resulting in a mean antibody concentration of 106 mg/L (19% increase, p=<0.05) and sequence V19 (SEQ ID No. 9) resulting in a mean antibody concentration of 118 mg/L (32% increase, p=<0.05). The data from stable cell lines are depicted in Figure 1. 20 When product quality was evaluated by SDS-electrophoresis no gross structural differences were observed between antibodies generated using the control or alternative signal sequences. Assessment of product quality by mass spectroscopy demonstrated that the antibodies generated using the alternative signal sequences were of the expected mass compared to the control suggesting that they were likely to be processed appropriately. 25 This work demonstrates that different signal sequences function with varying efficiency which indicates that the choice of signal sequence is likely to be a key contributor towards optimal antibody expression. In order to determine if this sequence would lead to an increase in antibody concentration 30 throughout the cell line construction process, constructs were generated encoding the IgG1/kappa antibody version of cB72.3. The constructs used either the gene-optimised genes fused to DNA sequence encoding the signal sequence of SEQ ID No. 9 (V 19) (pcB72.3IgG1V19) or antibody derived signal sequences routinely used (pcB72.3IgG1).
WO 2008/148519 PCT/EP2008/004413 23 These constructs were stably transfected into CHOKI SV cells. Transfectants were screened in 96-well plates and the high ranking cell lines expanded into 24-well plates. On reaching confluence, the top-60 producers were progressed into T25 flasks and then adapted to suspension in shake-flasks. Productivity was assessed in both batch and fed 5 batch shake-flask cultures and product quality analysed using culture supernatant from 14 day fed-batch overgrow cultures. In fed-batch cultures, the construct employing the signal sequence of SEQ ID No. 9 demonstrated a 6% increase in mean antibody concentration compared to controls 10 (p=0.08). Product quality assessment revealed no notable differences between cultures transfected with the control or constructs with the signal sequence of SEQ ID No. 9 (V 19) when analysed by SDS-electrophoresis, IEF, oligosaccharide analysis. Electrospray mass spectroscopy was used to show that the mass of the antibody generated using the V19 sequence was the same as that when generated using the control construct suggesting that 15 the V19 sequence was correct processed during antibody secretion.. In fed-batch bioreactor cultures employing 10L labscale bioreactors, Sixteen 10 litre laboratory-scale bioreactor cultures were performed. The cell lines reached a wide range of maximum viable cell concentrations. The cell lines containing the signal sequence of 20 SEQ ID No. 9 (V19) reached between 9.6 and 17.9 x 106 cells/mL and the control signal sequence cell lines reached between 8.5 and 19.1 x 106 cells/mL. The average time integral of viable cell concentration (IVC) for the 'new signal sequence' cultures was 2395 x 106 cells.h/mL whereas the IVC was 2511 x 106 cells.h/mL for the control cell lines. The average product concentration at harvest was 2870.3 mg/L for the cell lines with signal 25 sequence V19 (SEQ ID NO. 9) and 2577.1 mg/L for the control cell lines. The bioreactor cultures were harvested on day 15. Based on the laboratory-scale bioreactor cultures it was concluded that the V19 containing cell lines resulted in an increase in mean antibody concentration of 11%, however the 30 difference was not statistically significant due to the wide range of productivities observed.